The present invention relates to a non-invasive method and apparatus for detecting biological activities in a fluid specimen, such as blood, urine or sputum, where the specimen and a culture medium are introduced into sealable containers and are exposed to conditions enabling a variety of metabolic, physical, and chemical changes to take place in the presence of microorganisms in the sample. The biological activity being detected by a variety of chemical sensors that are based on changes in fluorescence lifetime and/or intensity.
Usually, the presence of microorganisms such as bacteria in a patient's body fluid, particularly blood, is determined using blood culture vials. A small quantity of blood is injected through a sealing rubber septum into a sterile vial containing a culture medium. The vial is incubated at a temperature conducive to bacterial growth, e.g., 37.degree. C., and monitored for such growth.
Common visual inspection involves monitoring the turbidity of the liquid suspension. Known instrumental methods detect changes in the CO.sub.2 content in the head space of the culture bottles, which is a metabolic by-product of the bacterial growth. Monitoring the CO.sub.2 content can be accomplished by conventional methods, including radiochemical, infrared absorption at a CO.sub.2 spectral line, or pressure/vacuum measurement. These methods, however, require invasive procedures which can result in cross-contamination between vials.
Recently, novel non-invasive methods have been developed which use chemical sensors inside a vial. Such sensors often respond to changes in the CO.sub.2 concentration by changing their color or by changing their fluorescence intensity (see, e.g., U.S. Pat. No. 4,945,060). The outputs from these sensors are based upon light intensity measurements. This means that errors may occur, particularly if the light sources used to excite the sensors, or the photodetectors used to monitor intensities, exhibit aging effects over time.
In known automated non-invasive blood culture systems, individual light sources, individual spectral excitation and emission filters, and individual photodetectors are arranged adjacent to each vial. Such arrangements result in certain station sensitivity variations from one-vial to the next. Due to the fact that most known blood culture sensors generate only a moderate contrast ratio in the measured photocurrent during bacterial growth, extensive and time-consuming calibration procedures and sophisticated detection algorithms are required to operate these systems. Moreover, light sources, spectral filters, and photodetectors with extreme narrow specification tolerances must be utilized.
Even if it would be possible to equalize all vial stations, certain variations in the vial shape would still remain and affect the measured fluorescence photocurrent. In fluorescence intensity-based sensor arrangements, any drift in the excitation source intensity, any change in the photodetector sensitivity, or any dust contamination on an optical surface will also cause a change in the measured photocurrent. Therefore, the long-time stability of such an instrument would be poor and frequent re-calibration would be required.
The disadvantage of intensity-based sensor arrangements can be overcome by utilizing fluorescent sensors that change their fluorescence lifetime, wherein intensity measurement is replaced with time parameter measurement and intensity changes have no impact on the sensor output signal. Many chemical sensor materials are known that change their fluorescence lifetime with changing oxygen concentration, pH, carbon dioxide concentration, or other chemical parameters (see, e.g., G.B. Patent No. 2,132,348).
A change in sensor fluorescence lifetime is commonly monitored by applying a well-known phase shift method (see, e.g., U.S. Pat. No. 5,030,420), wherein the excitation light is intensity-modulated. That method results in an intensity-modulated fluorescence emission that is phase-shifted relative to the excitation phase. Phase shift angle, .theta., being dependent on the fluorescence lifetime, .tau., according to the equation: EQU tan .theta.=.omega..tau. (1)
where .omega.=2.pi.f, is the circular light modulation frequency.
An inspection of equation (1) reveals that the phase shift method allows for maximum resolution, d.theta./d.tau., under the condition .omega..tau.=1. Unfortunately, almost all known pH- or carbon dioxide-sensitive fluorophores have decay times in the range 5 ns to 500 ps. In other words, light modulation frequencies, f=1/2.pi..tau., in the range 32 MHz to 320 MHz would be required.
It is possible to accomplish light intensity modulation at such high frequencies, however, this would require acousto-optic or electro-optic modulators which are only efficient in combination with lasers. Moreover, detecting the modulated fluorescence light would require highly sensitive high-speed photodetectors, such as micro channel-plate photomultipliers, which are rather expensive. Consequently, all commercial automated blood culture systems are based on intensity monitoring, and none utilize time-resolved fluorescent carbon dioxide sensors.